Probing plasma membrane microdomains in cowpea protoplasts using lipidated GFP-fusion proteins and multimode FRET microscopy.

Summary Multimode fluorescence resonance energy transfer (FRET) microscopy was applied to study the plasma membrane organization using different lipidated green fluorescent protein (GFP)-fusion proteins co-expressed in cowpea protoplasts. Cyan fluorescent protein (CFP) was fused to the hyper variable region of a small maize GTPase (ROP7) and yellow fluorescent protein (YFP) was fused to the N-myristoylation motif of the calcium-dependent protein kinase 1 (LeCPK1) of tomato. Upon co-expressing in cowpea protoplasts a perfect co-localization at the plasma membrane of the constructs was observed. Acceptor-photobleaching FRET microscopy indicated a FRET efficiency of 58% in protoplasts co-expressing CFP-Zm7hvr and myrLeCPK1-YFP, whereas no FRET was apparent in protoplasts co-expressing CFP-Zm7hvr and YFP. Fluorescence spectral imaging microscopy (FSPIM) revealed, upon excitation at 435 nm, strong YFP emission in the fluorescence spectra of the protoplasts expressing CFP-Zm7hvr and myrLeCPK1-YFP. Also, fluorescence lifetime imaging microscopy (FLIM) analysis indicated FRET because the CFP fluorescence lifetime of CFP-Zm7hvr was reduced in the presence of myrLeCPK1-YFP. A FRET fluorescence recovery after photobleaching (FRAP) analysis on a partially acceptor-bleached protoplast co-expressing CFP-Zm7hvr and myrLeCPK1-YFP revealed slow requenching of the CFP fluorescence in the acceptor-bleached area upon diffusion of unbleached acceptors into this area. The slow exchange of myrLeCPK1-YFP in the complex with CFP-Zm7hvr reflects a relatively high stability of the complex. Together, the FRET data suggest the existence of plasma membrane lipid microdomains in cowpea protoplasts.

[Comparison of the efficacy of two different iron supplements for anemia prevention in piglets]. / Vergelijkend onderzoek naar de werkzaamheid van twee verschillende ijzersupplementen voor anemiepreventie in biggen.

In a randomized, confirmatory study performed between July and October 2000 the efficacy of two iron products in preventing iron deficiency anaemia was compared. A total of 102 newborn piglets from ten litters were treated intramuscularly with 200 mg iron as iron dextran per ml, or 200 mg iron as gleptoferron per ml. For true comparison, piglets within a litter of a sow were subdivided into pairs on the basis of birth weight (one pair of the two heaviest piglets, et cetera). Within a pair, treatment with the iron supplements was randomly allocated. One group of piglets was injected at an age of 1 day (experiment 1) and the other group of piglets was injected at an age of 3 days (experiment 2). The piglets were weighed and blood samples were taken at an age of 18 days (experiment 1) or at an age of 19 days (experiment 2). Average daily weight gain and haemoglobin concentrations of both treatment groups were compared. Both products were very effective in preventing anaemia. No significant differences could be found between the two formulations. It can be concluded that iron-dextran and gleptoferron can be used with similar effect for anaemia prevention in piglets.